To study the effects of the RAS on MPP⁺-induced neurotoxicity in vitro, primary rat E15 VM cultures were grown for 6 days and then treated with MPP⁺ (1–100 μM) for 48 hrs. MPP⁺-treated cultures showed a dose-dependent loss of TH immunoreactive DA neurons, with a statistically significant loss of 15.9 ± 3% to 71.8 ± 6% over a dose range of 1 to 100 μM (Fig. 2).

In an effort to determine whether AT receptor activation affects the resistance of DA neurons to MPP⁺ toxicity, VM cultures were treated with Ang II (100 nM) prior to the addition of MPP⁺ (1–100 μM). Select VM cultures were also pretreated with the AT₁ receptor antagonist losartan (1 μM) and/or the AT₂ receptor antagonist PD123319 (1 μM), prior to the addition of Ang II (Fig. 2). TH⁺ neuronal counts were determined 48 hrs after the addition of MPP⁺. MPP⁺ dose response conditions were analyzed by one-way ANOVA followed by a Newman-Keuls post-hoc test. This analysis showed that MPP⁺-treated VM cultures pretreated with Ang II (100 nM), in the presence of losartan (1 μM), had a statistically significant reduction in DA neuronal loss when compared to MPP⁺ exposed VM cultures pretreated with Ang II alone or in the presence of PD123319 (Fig. 2). VM cultures treated with Ang II and losartan showed a 71.6 ± 6% and 48.3 ± 10% reduction in MPP⁺-induced DA neuronal loss when compared to 5 μM and 10 μM MPP⁺ alone treated VM cultures, respectively (Fig. 2). In combination, both AT receptor antagonists in the presence of Ang II (100 nM) did not significantly alter MPP⁺ toxicity. Ang II alone or in the presence of PD123319, did not provide significant neuroprotection (Fig. 2). Both AT receptor antagonists, in the absence of exogenous Ang II, also did not affect MPP⁺ toxicity (data not shown). In addition, VM cultures treated with Ang II (100 nM) and losartan (1 μM) 15 to 60 min after the addition of MPP⁺ (10 μM) and assayed 48 hrs later, were not neuroprotected against MPP⁺ (data not shown). This suggests that modification of downstream signaling of the angiotensin pathway is required prior to neurotoxin exposure.

Since downstream signaling of Ang II is dependent on specific AT receptor distribution, we began examining AT₁ and AT₂ receptor expression in DA neurons in vitro. While the majority (95%) of TH⁺ neurons expressed the AT₁ receptor using two different antibodies (Abcam and Santa Cruz) (Fig. 3a), only 65% of the TH⁺ neurons expressed the AT₂ receptor (detectable only with Abcam antibody) (Fig. 3b). Because of the difference in AT₁ and AT₂ receptor expression in DA neurons, the AT receptor profiles of other VM culture cell types were subsequently examined.

AT₁ receptors were co-expressed in cells immunopositive for nestin (neural progenitors), GFAP (astrocytes), NeuN and MAP2 (neurons) (Fig. 3a). AT₂ receptors were expressed in nestin, GFAP, NeuN and MAP2 stained cells (Fig. 3b). Approximately half of all cells showed double labeling for both AT receptors (Fig. 3c).

In an effort to determine specificity of the antibodies used for the detection of the AT₁ and AT₂ receptors we performed western immunoblot analysis of these receptors in N27 dopaminergic cells, which were derived from immortalized E12 VM. As seen in figure 4, both antibodies detect the AT_1A and the AT_1B and AT₂ receptors in these dopaminergic cells.

To better understand the role of the brain-RAS in preventing DA neuronal loss in vivo, we determined the AT receptor expression profile of TH⁺ cells of the SNpc of adult male C57BL/6 mice by immunohistochemistry. As seen in figure 5, TH⁺ neurons in the intact SNpc expressed a similar AT receptor profile to that found in VM cultures, with the majority of TH⁺ cells expressing the AT₁ receptor (95%), but the AT₂ receptor was expressed in fewer SNpc dopamine neurons (45%); detectable only with the Abcam antibody (Fig. 5). Because of the inherently semi-quantitative nature of the immunohistochemical analysis, we have also employed an alternative technique for assaying the AT₂ receptor expression by determining the levels of Agtr2 mRNA by real-time RT-PCR, in TH⁺ neurons of the SNpc and ventral tegmental area (VTA), isolated by laser capture microdissection (LCM). Low levels of Agtr2 mRNA were detected in these cells and there was no significant difference between TH⁺ neurons from the SNpc and the VTA (Fig. 6). Animal and cell culture treatments with MPTP or MPP⁺ did not change AT receptor immunoreactivity to any detectable level (data not shown). In addition, we did not observe any differences in the AT receptor profile between TH⁺ neurons of the SNpc and TH⁺ neurons of the VTA (data not shown), indicating that previously reported lower susceptibility to MPTP of the TH⁺ neurons from the VTA is not likely due to a difference in AT receptor distribution.

A severe loss (61.8 ± 10%) of DA neurons was observed in the SNpc of MPTP-treated mice compared to control mice treated with saline (Fig. 7). By contrast, the number of DA neurons lost in MPTP-treated mice receiving losartan was only 25.0 ± 16%, when compared to saline-treated mice. These results show that pretreatment and daily dosing with losartan can reduce MPTP-induced DA neuronal loss in the SNpc by 60% (p < 0.05). In an effort to confirm that the injected MPTP regimen induced DA neuronal loss and not just a down-regulation of TH immunoreactivity, we determined the total number of cells in the SNpc by Nissl staining. Nissl staining of the SNpc revealed a total cell loss of 54.1 ± 1% in MPTP-treated mice, demonstrating that the MPTP regimen resulted in DA neuronal cell loss (Fig. 7). MPTP-treated mice receiving losartan had a 44.9 ± 10% loss of total cells, which was not statistically different from the cell loss observed in mice receiving only MPTP. In mice treated with losartan alone, the numbers of DA neurons or Nissl-stained cells remained unaltered when compared to saline control mice (Fig. 7). Our results show that the AT₁ receptor antagonist, losartan, reduces MPTP-induced DA neuronal loss in the SNpc of C57BL/6 mice.

Significant differences between treated animal groups were also observed when measuring striatal TH⁺ immunostaining densities. Mice treated with MPTP showed a significant 70.6 ± 7% decrease in striatal TH⁺ staining when compared to saline control animals (Fig. 8). Daily treatment with losartan significantly reduced the loss of striatal TH immunoreactivity in the striatum of MPTP-treated mice by 53.2 ± 8% when compared to MPTP-alone treated mice (Fig. 8). Animals treated with losartan alone showed no significant change in TH immunoreactivity when compared to saline control animals (Fig. 8). Our results demonstrate that inhibition of the AT₁ receptor with losartan significantly reduces MPTP-induced denervation of striatal nerve terminals.

The possibility that losartan may interfere with brain uptake or metabolism of MPTP was examined in the SN and striatum of mice injected with MPTP. Sixty minutes after the last MPTP injection, the SN and striatum were dissected and MPP⁺ levels were determined through HPLC. No differences in nigral and striatal MPP⁺ levels were observed regardless of whether MPTP-injected mice were treated with losartan or not (Table 1). These findings highlight the fact that losartan does not interfere with brain uptake or metabolism of MPTP.

To rule out the possibility that losartan-mediated neuroprotection was a result of inhibition of the dopamine transporter (DAT) and subsequent reduction of MPP⁺ entry into DA neurons, VM cultures were treated with losartan (1 and 10 μM) and DA uptake levels were determined. Losartan (1 and 10 μM) did not alter DA uptake in VM cultures (Table 1). This demonstrates that losartan does not interfere with the function of the DAT.

In an effort to determine if losartan reduces blood pressure (BP) levels in C56BL/6 male mice, we took arterial BP measurements of mice treated with saline or losartan. In order to maintain constant BP levels, losartan was administered in drinking water at a dose of 0.18 mg/mL. Table 2 shows the systolic and diastolic measurements after this dosing regimen. Losartan alone reduced mean BP by 24 ± 3% when compared to saline treated controls (Table 2).

Rat ventral mesencephalic (VM) tissues from E15 Sprague Dawley embryos were dissected into Ca⁺²/Mg⁺² – free Hank's buffered salt solution (HBSS), mechanically dispersed and centrifuged 49. The cells were resuspended in F12 medium with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Sigma). Viability was determined using trypan blue exclusion. Cells were seeded at a density of 6 × 10⁴ viable cells/cm² on polyethylenimine (1 μg/ml) pre-coated 96- and 12-well culture plates or 8-chambered glass slides and incubated for 6 days at 5% CO₂/37°C. Half of the medium was changed every 3–4 days. After 6 days of growth, primary VM cultures consist of approximately 55% neurons, 45% type 1 astrocytes and <1% other cell types, as determined by immunohistochemical analysis 50. Dopaminergic (TH⁺) neurons represented 2–5% of total cells in VM cultures.

VM cultures were exposed to the parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium (MPP⁺) (1–100 μM) (Sigma) for 48 hr at 37°C and then processed for analysis. In Ang II-treated cultures, the octapeptide (1–100 nM) (Sigma) was added either 30 min prior or 15, 30 or 60 min after MPP⁺ treatment. In an effort to determine AT receptor subtype specificity, VM cultures were pretreated with 2-n-Butyl-4-chloro-5-hydroxy-methyl-1-[(2'-(1H)-tetrazol-5-yl)biph enyl-4-yl)methyl]imidazol potassium salt (DuP 753, losartan) (1 μM) (AT₁ receptor antagonist) (Du Pont/Merck, Wilmington, DE) or PD123319 (1 μM) (AT₂ receptor antagonist) (Sigma) or both, 15 min prior to the addition of Ang II 1920. Cells were exposed to the treatment agents for the duration of the experiment. VM cultures were then washed with PBS and fixed by the addition of 4% paraformaldehyde until analyzed.

Eight-week-old male C57BL/6 mice (Charles River Laboratories, Wilmington, MA) were used. Mice (n = 4–5 per group) received subcutaneous (200 μl) injections of 2-n-Butyl-4-chloro-5-hydroxy-methyl-1-[(2'-(1H)-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazol potassium salt (DuP 753, losartan) (90 mg/kg) (100 mg tablets, Du Pont/Merck, Wilmington, DE; dissolved in saline and filtered through a 0.2 μm filter) daily for 16 days, beginning two days prior to the first MPTP injection (Fig. 1). It is expected that the current dosing of losartan should result in greater than 60% inhibition of AT₁ receptors in the brain 51. Control animals received saline injections. Losartan and saline control treated mice received four intraperitoneal injections of MPTP-hydrochloride (20 mg/kg of free base; Sigma) in saline at 2-hour intervals on day 3 followed by a single intraperitoneal injection of MPTP-hydrochloride (20 mg/kg) on day 9, and were sacrificed 7 days after the last MPTP injection 52. Control mice received saline injections. This protocol is in accordance with the NIH guidelines for use of live animals and was approved by the Institutional Animal Care and Use Committee of the University of Colorado at Denver and Health Sciences Center.

For immunohistochemical analysis, animals were perfused with 20 ml of saline followed by 20 ml of freshly made 4% paraformaldehyde. Brains were then removed and stored in 4% paraformaldehyde until analyzed. Fixed VM cultures or 40 μm brain sections were incubated with an appropriate primary antibody overnight at 4°C, followed by 1 hr incubation with a secondary antibody at room temperature. To identify DA neurons, a polyclonal (rabbit) anti-TH antibody (1:500) (Pel-Freez, Rogers, AR) was used. TH⁺ cells were visualized by nickel-enhanced DAB staining (Pierce) using a Vectastain kit (Vector laboratories, Burlingame, CA) or by immunofluorescence using an appropriate secondary antibody. Different cell types in the cultures were identified with a polyclonal (mouse) anti-glial fibrillary acidic protein (GFAP) (1:200) for astrocytes, a polyclonal (mouse) anti-nestin (1:400) for neural progenitors, and a polyclonal (mouse) anti-microtubule-associated protein 2 (MAP2) (1:200) or a polyclonal (mouse) anti-neuronal nuclear protein (NeuN) (1:200) for neuronal cell types (Chemicon, Temecula, CA). To determine AT receptor subtypes, a commercially available polyclonal (rabbit) anti-AT₁ (N-10) (1:50) (Santa Cruz Biotechnology, Inc), polyclonal (goat) anti-AT₂ (H-143) (1:50) (Santa Cruz Biotechnology, Inc), polyclonal anti-AT₁(rabbit) (18801) (1:200) (Abcam), polyclonal (rabbit) anti-AT₂ (C-18) (1:100) (Santa Cruz Biotechnology, Inc) or a polyclonal anti-AT₂ (rabbit) (19134) (1:200) (Abcam) antibodies were used 1920. Secondary antibodies used were Alexa 488-conjugated (donkey) anti-goat IgG (1:1000), Alexa 488-conjugated (donkey) anti-rabbit IgG (1:1000), Alexa 594-conjugated (donkey) anti-rabbit IgG (1:1000) and Alexa 594-conjugated (donkey) anti-mouse IgG (1:1000) (Molecular Probes, Eugene, OR). Total cells were visualized by Hoechst stained nuclei. Double and triple staining was done consecutively. Stained cultures were visualized by light and/or fluorescence microscopy using Zeiss or Nikon microscopes. For AT receptor profile counts in vitro, a total of 700 TH⁺ cells co-labeled for AT₁ or AT₂ receptor were counted from a minimum of 3 fields. For the in vivo AT receptor profile, TH⁺ cells co-labeled for AT₁ or AT₂ receptor were counted from the SNpc of 3 coronal sections. To determine the number of total cells in the SNpc, selected sections were defatted, rehydrated through descending alcohol concentrations, stained in cresyl violet acetate for 1 minute followed by acetic formalin, then dehydrated and cleared in xylene before coverslipping in Permount. Quantification of VM culture TH⁺ cells' survival was done by a blinded observer who counted all the positively stained cells having a distinct nucleus and visible neurites in the entire well. Each experimental condition in vitro represents 4–6 independent experiments.

HPLC with UV detection (wavelength = 295 nm) was used to measure striatal MPP⁺ levels 56. Mice were injected subcutaneously (n = 3) daily with losartan (90 mg/kg) or saline for 3 days. On day 3, mice were injected 4 times every 2 hours intraperitoneally with saline or MPTP-hydrochloride (20 mg/kg). Sixty minutes after the last MPTP injection mice were sacrificed and the brains were immediately removed. The substantia nigra (SN) and striatum were dissected out on ice and rapidly frozen on dry ice and stored at -80°C until analysis. On the day of the assay, SN and striatum were prepared by sonicating the tissue samples in 9% (wt/vol) of 5% trichloroacetic acid (Sigma) containing 5 μg/ml of 4-phenylpyridine (Sigma), as an internal standard. After centrifugation, 50 μl of supernatant were injected onto a cation-exchange C18 column (Alltech). The mobile phase consisted of 89% (v/v) 50 mM KH₂PO₄ (pH 3 adjusted with H₃PO₄) and 11% (v/v) acetonitrile. The flow rate was 1.5 ml/min.

Dissociated mesencephalic neurons were grown as described above. The neurons were rinsed and then assayed at 37°C in Krebs-Ringer HEPES buffer (KRH; 120 mM NaCl, 4.7 mM KCl, 2.2 mM CaCl₂, 1.2 mM Mg SO₄, 1.2 mM KH₂PO₄, 10 mM glucose, 10 mM HEPES, pH 7.4) supplemented with 10 μM pargyline, 10 μM ascorbic acid, and 10 μM catechol. Assays (1 ml) included 100 nM [³H] DA. Nonspecific [³H] DA accumulation was determined in the presence of 1 mM (-) cocaine hydrochloride. After 10 min of incubation at 37°C, uptake was terminated by quickly washing the neurons three times with 1 ml of ice-cold KRH. Neurons were then solubilized in 0.5 ml of 3% trichloroacetic acid for 60 min with gentle shaking. Accumulated [³H] DA was determined by liquid scintillation counting (n = 3) 57.

Losartan (0.18 mg/mL) was administered through the drinking water and tail artery blood pressures were recorded non-invasively from awake animals. Animals were placed in a restraining tube heated to 37°C, and a pressure cuff and piezoelectric transducer were secured to the rostral base of the tail (PowerLab, AD Instruments). Animals were conditioned for ten to fifteen minutes by inflating the pressure cuff to 200 mmHg once every minute. Post-conditioning pulse and cuff pressure recordings were simultaneously acquired for up to 45 min at one reading per minute. Motion artifact detected by the pulse transducer during cuff deflation served as grounds for elimination of data points. Systolic and diastolic pressures were determined offline (Chart, AD Instruments). Systolic pressure was defined as cuff pressure upon first detection of pulse during deflation. Diastolic pressure was defined as cuff pressure when the pulse reading resumed normal amplitude (n = 4, >32 readings per animal).

Substantia nigra sections were immunostained for TH or stained with cresyl violet as described above. The SN in C57BL/6 mice measures approximately 1.7 mm in rostrocaudal extent. Based on a comparative mouse brain atlas 58, the SN is completely contained between the coronal planes defined between -2.40 mm and -4.20 mm from bregma. A total of 54 (40 μm) sections were cut on a cryostat and separated into three section pools using every third section per pool. The three section pools contained the entire SN. Due to tissue shrinkage the actual thickness of fixed and stained tissue was on average 21 μm. TH-immunoreactive SNpc neurons were estimated using an unbiased stereological method according to the optical fractionator principle 596061 using a Leica DMRB microscope with motorized stage running BioQuant Nova Prime Revision 3.0 (BioQuant Image Analysis Corporation, Nashville, TN) on a 75 μm × 75 μm grid with a dissector size of 50 μm × 50 μm. The contour outlines and landmarks of the SNpc, which comprise the A9 cell group, were drawn on an image captured with a 5× objective. Using a 100× objective and starting at the top of the section with the top plane of the cells in focus, the z-plane guards were determined by excluding 4 μm from the surfaces and only the TH⁺ profiles that came into focus within the counting frame thickness (13 μm) between the guard zones were counted. Care was taken to ensure that the top and bottom forbidden planes were not included in the analysis. Adjacent sections were stained with cresyl violet acetate and cells in the SNpc were counted in a similar manner as with the TH⁺ immunostained sections, with the exception that large nuclear Nissl stained cells were counted instead of TH⁺ cells. Because Nissl staining was done separately from TH immunostaining, it is possible that non-dopaminergic neurons and glia were also included in the total cell counts. Because of the careful demarcation of the SNpc (Fig. 7a), the majority of large nuclear Nissl stained cells in the selected area are likely to be neurons of the A9 cell group. All stereological counts were done blindly to the treatment conditions. A coefficient of error (CE) of <0.02 was accepted (Table 3), for TH (n = 4–5 animals/group) and Nissl stained cells (n = 3 animals/group). Counts are represented as mean ± SEM.

Twelve-week-old male C57BL/6 mice were euthanized using CO₂ asphyxiation. The brains were removed and immediately frozen in isopentane on dry ice. Brains were then stored at -80°C until analysis. Brains were then cut on a cryostat and 10 μm coronal sections were mounted on uncoated slides and stored at -80°C. Slides were allowed to briefly reach room temperature and were stained for TH using a rapid staining protocol to minimize mRNA degradation. Briefly, slides were fixed in acetone (40 seconds), washed briefly 3 times in phosphate buffer (PB; pH = 7.4), followed by a 5 min incubation with a mouse-anti-TH antibody (Sigma, 1:1000) diluted in PB. Slides were washed 3 times with PB, followed by 5 minute incubation with a Cy3-conjugated goat anti-mouse IgG (Jackson Labs, 1:500) diluted in PB. Slides were washed twice in PB and then dehydrated in ethanol (50%, 70%, 95%, and 100%), (10 seconds each) and delipidated in xylene (40 seconds). Slides were then allowed to air dry. All procedures were carried out in an RNAse-free environment.

Tyrosine hydroxylase positive cells of the SNpc and VTA were visualized and dissected using a PixCell laser capture microscope under a 20× objective with an infrared diode laser (Arcturus Engineering, Santa Clara, CA). Approximately 500 TH positive cells per animal from the SNpc or VTA we isolated on a single LCM HS cap (Arcturuss Arcturus Engineering, Santa Clara, CA). RNAs were isolated using a PicoPure RNA Isolation kit (Arcturus, Mountain View, CA, USA) according to manufacturer's guidelines, and subjected to DNAse treatment (Qiagen, Valencia, CA, USA).

Atgr2, TH and GAPDH mRNA expression profiles were done by quantitative real-time PCR on TH⁺ neurons from the SNpc or VTA. Primers used are listed in table 4. The primers (18–22 mer) were designed using Primer3 62. These primer sets were designed to amplify small amplicons for candidate mRNAs ranging from 100–300 bp in size. First-strand cDNA synthesis was carried out on mRNA extracted with SuperscriptTM first-strand synthesis kit (Invitrogen Carlsbad, CA) according to the manufacturer's specifications. Real-time RT-PCR was carried out in a 96 well plate using a MyiQ iCycler (BioRad, Hercules, CA), and SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). A concentration curve with known concentrations of whole brain cDNA extracts from 12-week-old male C57BL/6 mice was used to calculate standard curves and quantitate the products. The final concentration of each transcript was calculated using the MyiQ2.0 software provided by BioRad.

The expression of AT₁ and AT₂ receptors was examined in an immortalized mesencephalic dopaminergic cell line, N27 63. N27 cells were thawed into RPMI 1640 media containing 10% FBS, 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were collected in lysis buffer (Cell Signaling Technology, Beverly, MA) with 1% SDS. After sonication, the lysate was centrifuged at 10,000 × g for 45 min at 4°C, and the proteins in the supernatant were separated on 4–12% Bis-Tris polyacrylamide gel. After transfer to PVDF membranes, the proteins were probed for AT₁ receptors (Santa Cruz polyclonal sc-1173 or Abcam polyclonal ab 18801, 1:500 dilution), AT₂ receptors (Santa Cruz polyclonal sc-9040 or Abcam polyclonal ab 19134, 1:500 dilution). Secondary, alkaline phosphatase-conjugated anti-rabbit (1:5000; Sigma) and anti-mouse (1:10,000, Jackson ImmunoResearch) antibodies were followed by Lumi-Phos (Pierce) for chemiluminescence detection. Blots were stripped and reprobed for β-actin (Sigma, 1:10,000) to confirm equal protein loading on the gel.

Data were analyzed using Statistica software. Statistical significance was determined by one-way ANOVA followed by post-hoc Newman-Keuls multiple comparison tests. Differences in mean values were considered significant at p ≤ 0.05. All data were obtained from at least three independent experiments and are represented as a mean ± SEM.